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Old 08-16-2017, 09:24 AM   #4
Location: United States

Join Date: May 2014
Posts: 40

Gotcha, so your control has worked well before (and is passing your standard QC) but isn't yielding anything in the context of your gradient touch-down PCR? What input mass are you using for your amplifications? And have your primers worked well in your hands in the past? Can you share your exact conditions for PCR (in your standard protocol and your gradient touch-down setup)?

I also use a highly processive/proofreading enzyme (NEB's Q5) for 16S PCR but I use the primers that were modified as part of the Earth Microbiome Project rather than Illumina's standard primers. Usually I think of the two-step PCR method as being more specific to the V3/4 locus, though -- my group had severe issues with mispriming at our standard annealing temperature in the past and we chalked it up to the one-step addition of adapter sequences. Can you share some gel images showing the products you're seeing besides your expected 550 bp band?

You may find this paper helpful for a comparison of primers and enzymes in the context of 16S PCR, by the way.
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